Despite the resurgence in interest in basic metabolic control, a large gap in knowledge concerns cell and context specific alteration and adaptation in each key pathway including the mTOR and AMPK pathways. In this proposal we will investigate a new pathway that links amino acid sensing with downstream alterations in T cell metabolism that have profound effects on T cell phenotype and function. The availability of essential amino acids has emerged as a central metabolic mechanism that regulates both T cell growth and the eventual T cell differentiation state. Antigen-presenting cells (APCs) regulate amino acid bioavailability to T cells via expression of inducible enzymes that degrade essential amino acids such as arginine and tryptophan, and have been hypothesized to restrict T cell activity and proliferation in immune microenvironments. The metabolic consequence to the T cells exposed to an amino acid-depleted microenvironment, and the downstream effects on T cell differentiation are at present poorly understood. We have discovered that arginine depletion has a central role in T cell metabolism that is directly relevant to in vivo diseases dominated by Th2 responses. To dissect the interplay between amino acid-depleting antigen presenting cells (APCs) and T cells we built a system where the APC, T cell and culture conditions can be manipulated by genetics and drugs that affect key metabolic checkpoints. Our approach has allowed us to evaluate metabolism in primary T cells undergoing APC-mediated arginine restriction, and can be used generally to investigate the consequences to both the T cell and APC for any amino acid. Our preliminary data suggests that arginine sensing by T cells involves an unanticipated mechanism that runs parallel to mTOR signaling to selectively shut down cholesterol/fatty acid biosynthesis but simultaneously maintain glycolysis. In this proposal we will investigate two elements of T cell metabolism. In Aim 1 we will define the hierarchy of signaling pathways activated by arginine starvation that leads to maintenance of glycolysis but ablation of cholesterol/fatty acid biosynthesis. Using genome-wide expression profiling of arginine-starved T cells compared to their dividing counterparts, we have discovered that the major downstream pathway responsive to arginine starvation is a shutdown of cholesterol/fatty acid biosynthesis that is completely reversible by exogenous arginine replacement. We will use genetic, biochemical and chemical methods to explore how the mTOR and AMPK and related pathways convert information about arginine levels into downstream metabolic decision points. In Aim 2 we will determine the interplay between key metabolic checkpoints including mTOR and AMPK that leads to helper T cell phenotypic plasticity. We have discovered that arginine-starved T cells change their fate to adopt characteristics of both Tregs and Th17 T cells. We will use innovative genetic approaches to track the genetic fate of T cells focusing on Treg-like cells marked with Foxp3-gfp. We will separate distinct T cell populations within arginine starved cultures based on Foxp3 expression and use these cells to understand how T cells use metabolic check points to make decisions about their phenotype. PUBLIC HEALTH RELEVANCE: Excessive T cell responses are a hallmark and driver of numerous autoimmune syndromes and diseases associated with disregulated immune responses. Our study focuses of a pathway that controls T cell proliferation and phenotype via sensing of amino acid depletion in microenvironments. We have found that amino acid sensing leads to metabolic pathway-driven changes in T cell fate. As such, our studies have wide implications for understanding and treating autoimmune syndromes.